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NeurobiologyIncrease of synaptic density and memory retention by a peptide representing the trophic domain of the amyloid ,t/A4 protein precursor (

Abstract. The growth of A-1 fibroblasts depends on exogenous amyloid ~/A4 protein precursor (APP), providing a simple bioassay to study the function of APP. Our preliminary study, testing the activity of a series of fragments derived from the secreted form of APP-695 (sAPP-695) on this bioassay, has shown that at least one of the active sites of sAPP-695 was localized within a 40-mer sequence (APP296-335, Kang sequence; Roch, J.-M., I. P. Shapiro, M. P. Sundsmo, D. A. C. Otero, L. M. Refolo, N. K. Robakis, and T. Saitoh. 1992. Z BioL Chem. 267:2214-2221. In the present study, to further characterize the growthpromoting activity of sAPP-695 on fibroblasts, we applied a battery of synthetic peptides on this bioassay and found that: (a) the sequence of five amino acids, RERMS (APP328-332), was uniquely required for the growth-promoting activity of sAPP-695; (b) the activity was sequence-specific because the reverse-sequence peptide of the active domain had no activity; and (c) the four-amino-acid peptide RMSQ (APP330-333), which partially overlaps the COOH-terminal side of the active sequence RERMS, could antagonize the activity of sAPP-695. Furthermore, a recombinant protein which lacks this active domain (APP20-591 without 306-335) did not promote fibroblast cell growth, suggesting that this domain is the only site of sAPP-695 involved in the growth stimulation. The availability of these biologically active, short peptides and their antagonists should prove to be an essential step for the elucidation of APP involvement in regulation of cellular homeostasis.

In various species, thyrotropin (TSH) is known to stimulate both differentiation and proliferation of thyroid follicle cells. This cell type has also been shown to express members of the Alzheimer amyloid precursor (APP) protein family and to release the secretory N-terminal domain of APP (sAPP) in a TSH-dependent fashion. In this study on binding to the cell surfaces, exogenously added recombinant sAPP stimulated phosphorylation mediated by mitogenactivated protein kinase and effectively evoked proliferation in the rat thyroid epithelial cell line FRTL-5. To see whether this proliverative effect of sAPP is of physiological relevance, we used antisense techniques to selectively inhibit the expression of APP and the proteolytic release of sAPP by cells grown in the presence of TSH. The antisense-induced inhibition was detected by immunoblot, immunoprecipitation, and immunocytochemical analyses. After the reduced APP expression and sAPP secretion, we observed a strong suppression of the TSH-induced cell proliferation down to 35%. Recombinant sAPP but not TSH was able to overcome this antisense effect and to completely restore cell proliferation, indicating that sAPP acts downstream of TSH, in that it is released from thyroid epithelial cells during TSH-induced differentiation. We propose that sAPP operates as an autocrine growth factor mediating the proliferative effect of TSH on neighboring thyroid epithelial cells.

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